ICAM-1, a member of the immunoglobulin (Ig) superfamily, is an inducible transmembrane glycoprotein constitutively expressed at low levels on vascular endothelial cells and on a subset of leucocytes (Dustin et al., J. Immunol., 137:245-54, 1986; Rothlein et al, J. Immunol., 137:1270-4, 1986; Simmons et al, Nature, 331:624-7, 1988). The primary ligands for ICAM-1 binding are the β2 integrins, LFA-1 and Mac-1, both of which are expressed on leukocytes (Marlin and Springer, Cell, 51:813-9, 1987; Diamond et al, J. Cell Biol., 111:3129-39, 1990). ICAM-1 serves multiple functions in the propagation of inflammatory processes, the best characterized being the facilitation of leukocyte migration from the intravascular compartment to the extravascular space at sites of inflammation (Butcher, Cell, 67:1033-6, 1991; Furie et al, Blood, 78:2089-97, 1991; Oppenheimer-Marks et al., J. Immunol., 147:2913-21, 1991). In addition, ICAM-1 also appears to provide an important second signal to T-lymphocytes during antigen presentation (Altmann et al., Nature, 338:512-4, 1989; Van Seventer et al., J. ImmunoL 144:4579-86, 1990; Kuhlman et al., J. Immunol., 146:1773-82, 1991). It also plays and important faciliatory roll in cytotoxic T-cell (Makgoba et al., Eur. J. Immunol., 18:637-40, 1988), natural killer cell (Allavena et al., Blood, 84:2261-8, 1994), and neutrophil-mediated (Ding et al., J. Immunol, 163:5029-38, 1999) damage to target cells.
In response to pro-inflammatory stimulators, including tumor necrosis factor-alpha (TNF-α) (To et al., Arthritis Rheum., 39:467-77, 1996; Beutler, Arthritis Rheum., 26:16-21, 1999), many cell types modulate the expression of ICAM-1 on their surface. Cellular adhesion molecules, including ICAM-1, are required for the migration of leukocytes and endothelial cells. Numerous studies have demonstrated an increase in ICAM-1 expression within involved tissues from patients suffering from a wide range of inflammatory diseases, including inflammatory bowel disease (IBD) (Jones et al., Gut, 36:724-30, 1995), rheumatoid arthritis (To et al., Arthritis Rheum., 39:467-77, 1996), celiac disease (Sturgess et al., Clin, Exp. Immunol. 82:489-92, 1990), IgA neuropathy (Nguyen et al., Am. J. Nephrol. 19:495-9, 1999), systemic lupus (Papa et al., Lupus, 8:423-9, 1999; Egerer et al., Lupus, 9:614-21, 2000), inflammatory dermatosis (Ackermann et al., Arch Dermatol Res., 290:353-9, 1998), and multiple sclerosis (Bo et al., J. Neuropathol. Exp. Neurol., 55:1060-72, 1996).
Anti-ICAM-1 monoclonal antibodies have demonstrated beneficial effects in a variety of animal models of disease, including pulmonary inflammation and asthma (Barton et al., J. Immunol. 143:1278-82, 1989; Wegener et al., Science, 247:456-9, 1990), allograft rejection (Cosimi et al., J. Immunol., 144:4604-12, 1990; Isobe et al., Science, 255:1125-7, 1993), nephritis (Flaming et al., Clin. Immunol. Immunopath., 64:129-34, 1992; Kawasaki et al., J. Immunol., 150:1074-83, 1993), ischemic injury (Ma et al., Circulation, 86:937-46, 1992; Kelly et al., Proc. Natl. Acad. Sci., USA. 91:812-6, 1994), inflammatory arthritis (ligo et al., J. Immunol., 147:4167-71, 1991), contact dermatitis (Scheynius et al., J. Immunol. 150:655-63, 1993), and colitis (Hamamoto et al., Clin. Exp. Immunol., 117:462-8, 1999). These animal models supported trials of inhibitors of either ICAM-1 function or expression in human disease.
ICAM-1 expression has been demonstrated during inflammatory bowel disease (IBD). In a mouse model of colitis induced by dextran sulfate, ICAM-1 expression is increased on the endothelium of colonic submucosal and tunica muscularis veins (Bennett, J. Pharm. Exp. Ther., 280:988-1000, 1997). Human ICAM-1 expression in Crohn's disease is increased in the lamina propria micorovasculature (both colonic and jejunal) (Sousa et al., Gut, 45:856-63, 1999), gut mononuclear cells (Bernstein et al., Clin. Immunopathol., 86:147-60, 1998), and apical portions of the colonic epithelium (Vanier et al., Am. J. Surg. Pathol., 24:1115-24, 2000). Tissue expression of ICAM-1 correlates with disease activity (Vanier et al., Clin. Exp. Immunol., 121:242-7, 2000).
Inflammatory bowel disease (IBD) refers to a group of diseases including both Crohn's disease and ulcerative colitis. The two diseases are often grouped together due to their similar pathogenesis and clinical manifestations. In the absence of invasive imaging studies, it is not possible to distinguish the two diseases which are often considered as a single disease in a number of publications and studies. Definitive diagnosis of either disease requires imaging studies such as endoscopy (either sigmoidoscopy or colonoscopy), double contrast barium enema, and computed tomography (CT) scan; combined with laboratory tests including complete blood counts to detect elevated leukocyte levels, erythrocyte sedimentation rates and serum albumin concentration.
Both diseases are chronic, relapsing/remitting inflammatory diseases of the gastrointenstinal (GI) tract. The regions of the GI tract that are most often affected by Crohn's disease are the small intestine and large intestine, also called the colon, including the rectum; however, Crohn's disease can affect the entire GI tract from the mouth to the anus. There may be single or multiple patches of inflammation. Ulcerative colitis affects only the large intestine. Inflammation and ulceration in ulcerative colitis are limited to the mucosal and submucosal layers, two innermost layers of the four layers of the large intestine. The inflammation and ulceration in Crohn's disease can extend through all layers of the intestinal wall in both the small and large intestines. Common symptoms of the diseases include diarrhea, abdominal pain, rectal bleeding and weight loss. Complications of Crohn's disease include intestinal abscesses, fistula, an abnormal passage leading from one portion of the intestine to another and permitting passage of fluids or secretions, and intestinal obstructions. Typically, the course of both diseases is intermittent, with disease exacerbations followed by periods of remission. However, ulcerative colitis may be a single event, or continuous with unrelenting symptoms.
Based on the overlap in the pathology and clinical manifestations, it is not surprising that available therapies for Crohn's disease and ulcerative colitis are substantially overlapping. A notable difference is the possibility of the use of enemas for the treatment of ulcerative colitis as the diseases areas are proximal to the rectum. This allows for topical application of a therapeutic agent. As Crohn's disease typically includes involvement of the small intestine, systemic treatment is typically required. Although there are many choices for therapeutic interventions in IBD, many have undesirable side effects that make them less than ideal for treatment of chronic disease.
For mild ulcerative colitis, orally or topically (i.e. enema) delivered aminosalicylates are typically the first line of treatment. The aminosalicylate class consists of agents that contain 5-aminosalicyclic acid (5-ASA), is one of the oldest anti-inflammatory compounds employed in IBD. For example, the 5-ASA sulfasalazine was developed in the 1930's for the treatment of rheumatoid arthritis, and its utility in the treatment of IBD was established in the 1970's (Anthonisen et al., Scan. J. Gastroenterol., 9:549-554, 1974). Used in high doses, 5-ASAs can induce remission in acute attacks. Although commonly used for maintenance therapy, 5-ASAs have not been demonstrated to be effective in maintaining remission.
Commonly used 5-ASA formulations include sulfasalazine, oral and topical mesalamine, olsalazine and balsalazide. Various formulations are modified to provide available active drug to the site of interest (e.g. small or large intestine). Side effects are not uncommon with 5-ASAs. Sulfasalazine has a relatively high toxicity (approximately one third of patients), associated with the sulfa group, including headache, nausea, dyspepsia and anorexia. Less common side effects include fever, rash, arthralgia, hemolysis, neutropenia, exacerbation of colitis, hypersensitivity reactions and nephrotoxicity. Mesalamine, olsalazine and balsalazide which do not contain sulfa groups result in substantially fewer side effects, but still can cause rash, headaches and fever. Other more severe side effects have also been reported.
Mesalamine enema has been implicated in the production of an acute intolerance syndrome characterized by cramping, acute abdominal pain and bloody diarrhea, sometimes headache, fever and a rash. While using mesalamine enema, some patients have developed pancolitis; however, the frequency was lower than with a placebo treated group. The extent of absorption of mesalamine from enema is largely dependent on retention time and therefore varies. Systemic exposure is substantially reduced by enema administration as compared to oral administration; however, 10 to 30% of the daily dose can be recovered in 24-hour urine collection suggesting that the systemic exposure is not insubstantial. Under clinical conditions, patients demonstrate plasma levels of 2 ug/ml, about two thirds of which was the N-acetyl metabolite, at 10 to 12 hours post-administration. (Physicians Desk Reference, 53rd Edition. 1999. Medical Economics Data, Montvale, N.J., pp 3126-7)
Corticosteroids are among the most effective agents for inducing remission in IBD attacks and are typically the second therapeutic option upon failure of treatment with 5-ASAs. The compounds are delivered first either orally or rectally, with or without concomitant administration of 5-ASAs. Upon failure of oral delivery, the compounds are administered intravenously. Ideally, corticosteroids are used for only a short course of treatment and tapered off upon remission of disease.
Corticosteroids commonly used for the treatment of IBD include prednisone, budesonide and hydrocortisone. The use of corticosteroids is limited by the number of severe and significant side effects associated with their use. Common side effects of short term use include insomnia, night sweats, mood changes and altered glucose metabolism. Prolonged maintenance therapy is typically reserved only for severe, refractory cases. Prolonged therapy can lead to adrenal atrophy, whereas abrupt cessation can cause adrenal insufficiency, hypotension, and even death. Other side effects include acne, abnormal fat deposition, excessive hair growth and osteoporosis. In Crohn's disease, corticosteroids can thwart the healing of fistula, exacerbating the disease state.
Individuals responding to oral or rectal corticosteroids are often placed on a maintenance dose of 5-ASA. However, some physicians provide no pharmacological interventions during periods of remission. Individuals who require therapy with intravenous corticosteroids are typically maintained on an immunosuppressive agent such as 6-mercaptopurine and/or azathioprine, in combination with a 5-ASA. Parenteral nutrition is typically considered with such severe disease. When the patient does not respond to the above therapies, the immunosuppressant cyclosporine may be administered in an attempt to avoid surgery to remove the section of diseased intestine.
Immunosuppressant interventions are not without undesirable side effects. 6-Mercaptopurine and azathioprine can cause fever, rash, nausea and headache, with more severe side effects including leucopenia, pancreatitis, severe infections and bone marrow suppression. Cyclosporine can have more severe side effects including paresthesias (abnormal sensations like burning or tingling), excessive hair growth, hypertension, tremor, renal insufficiency, headache and opportunistic infections.
Antibiotics, typically ciprofloxacin or metronidazole, are used as add on therapies to 5-ASA or corticosteroids, especially with patients with fistulizing or colonic disease. As with all of the other therapies, there are side effects of long term treatment with antibiotics.
Infliximab is currently the pharmacotherapy of last resort in IBD. It is a chimeric monoclonal antibody composed of 75% human and 25% mouse protein. Infliximab is an inhibitor of tumor necrosis factor-alpha (TNF-α), a potent inflammatory cytokine. The drug acts as a sink by binding both soluble and membrane bound TNF-α. By inhibiting an activator high in the inflammatory cascade, a number of inflammatory pathways can be inhibited. The drug is administered intravenously first for treatment and subsequently as a maintenance drug every eight (8) weeks as indicated on the product label. However, as it is a biological agent, an immune response can limit utility of the drug. Therefore, immunosuppressive agents are typically given in conjunction with infliximab maintenance therapy. As with all other therapies for IBD, there are substantial side effects of infliximab. TNF-α plays an important role in the eradication of neoplastic cells; therefore, its suppression can lead to opportunistic infections, malignancies and other complications, especially as a long term strategy (Brown et al., Arthritis Rheum., 46:3151-8, 2002; Lee et al., Arthritis Rheum., 46:2565-70, 2002; Nahar et al., Ann. Pharmacotherapy, 37:1256-65, 2003).
Surgical interventions are a method of treatment of IBD, not a cure. Due to the chronic nature of IBD and the relatively early age of onset, multiple surgeries can be required over the lifetime of patients who are not responsive to pharmacological interventions. Removal of short portions of the intestine is possible without substantial side effects. However, removal of larger or multiple segments of the intestine can result in short bowel syndrome in which individuals are unable to absorb nutrients. Removal of portions of the large intestine can result in the need for colostomy or other further surgical procedures. Therefore, surgery is not a preferred method of treatment of IBD.
Surgical interventions for the treatment of IBD can result in further disease. Upon complete removal of the colon, an ileal pouch may be constructed from the small intestine by the surgeon to allow removal of feces through the anus rather than requiring a permanent ostomy. Pouchitis is a non-specific inflammation of the ileal pouch which typically occurs within the first two years after reconstruction. Symptoms include steadily increasing stool frequency that may be accompanied by incontinence, bleeding, fever and/or a feeling of urgency. Of those who have ulcerative colitis, approximately 20 to 30 percent experience at least one episode. Antibiotics can be sufficient to treat pouchitis; however, other more aggressive therapies similar to those used in IBD are required.
Antisense oligonucleotides offer an ideal solution to the problems encountered in prior art approaches. They can be designed to selectively inhibit expression of a given nucleic acid or protein, and avoid non-specific mechanisms of action by interacting with a nucleic acid target based on nucleotide sequence, allowing for the inhibition of a specific isoform of a family of similar protein structure or activity. A complete understanding of target mechanisms or macromolecular interactions is not needed to design specific inhibitors.
Human ICAM-1 is encoded by a 3.3-kb mRNA (SEQ ID NO 1) resulting in the synthesis of a 55,219 dalton protein. ICAM-1 is heavily glycosylated through N-linked glycosylation sites. The mature protein has an apparent molecular mass of 90 kDa as determined by SDS-polyacrylamide gel electrophoresis (Staunton et al., Cell, 52:925-933, 1988). ICAM-1 is a member of the immunoglobulin (Ig) superfamily. It contains five immunoglobulin-like domains at the amino terminus, followed by a transmembrane domain and a cytoplasmic domain. The primary binding site for LFA-1 and rhinovirus are found in the first immunoglobulin-like domain. However, the binding sites appear to be distinct (Staunton et al., Cell, 61:243-354, 1990).
ICAM-1 exhibits a broad tissue and cell distribution, and may be found on white blood cells, endothelial cells, fibroblast, keratinocytes and other epithelial cells. The expression of ICAM-1 can be regulated on vascular endothelial cells, fibroblasts, keratinocytes, astrocytes and several cell lines by treatment with bacterial lipopolysaccharide and cytokines such as interleukin-1, tumor necrosis factor, gamma-interferon, and lymphotoxin (See, e.g., Frohman et al., J. Neuroimmunol., 23:117-124, 1989).
A series of oligonucleotides were tested for the ability to inhibit the expression of human ICAM-1 (SEQ ID NO 1) using both in vitro and in vivo experiments (see e.g., U.S. Pat. No. 5,514,788). From these experiments, the oligonucleotide ISIS 2302 (SEQ ID NO 2) which is targeted to nucleotides 2114 to 2133 of human ICAM-1 was selected for further development.
ISIS 2302 is a 20-base phosphorothioate oligodeoxynucleotide designed to specifically hybridize to a sequence in the 3′-untranslated region of the human ICAM-1 mRNA. Studies strongly suggest that ISIS 2302 functions by specifically binding to the ICAM-1 mRNA resulting in cleavage of the mRNA by the enzyme RNaseH1 (Crooke, Biochim. Biophys. Acta., 1489:31-44, 1999), one of a ubiquitous family of RNaseH nucleases. However, the method of the invention is not limited by the mechanism of action of ISIS 2302.
Phosphorothioate modification of the oligodeoxynucleotide, by substituting a sulfur molecule for a non-bridging oxygen molecule in each phosphodiester linkage, significantly increases exonuclease resistance relative to unmodified DNA and prolongs the drug half life (Geary et al., Anti-Cancer Drug Design, 12:383-94, 1997). Phosphorothioate oligonucleotides are only minimally antigenic, non-cytotoxic and well tolerated, and their pharmacokinetic and pharmacodynamic properties are well characterized (see e.g., Butler et al., Lab. Invest., 77:379-88, 1997; Mirabelli et al., Anti-Cancer Drug Des., 6:647-61, 1991)
In addition to phosphorothioate backbone modifications, a number of other possible backbone, sugar and other modifications are well known to those skilled in the art and are discussed in the parent applications on which this application is based.
Antisense oligonucleotides targeted to ICAM were shown to be effective in mouse and rat models of inflammatory bowel disease when systemically administered by subcutaneous or intraperitoneal injection (see, e.g., Bennett, U.S. Pat. No. 5,514,788, example 20; and U.S. Pat. No. 6,096,722, example 29, respectively). These data were the foundation for the development of clinical trials for the treatment of Crohn's disease, pouchitis and ulcerative colitis in humans. In the Crohn's trial, ISIS 2302 (Alicaforsen) was administered systemically to individuals in placebo controlled studies. Phase II studies suggested efficacy of intravenously administered ISIS 2302 in some subsets of patients (Yacshyn et al., Gut, 51:30-36. 2002; Yacyshyn et al., Aliment. Pharmacol. Ther., 16:1761-70. 2002). In two subsequent Phase III trials, intravenously administered ISIS 2302 was found to be ineffective in treating moderate to severe Crohn's disease patients based on clinical remission and disease activity index scores (Chey et al., Gastroenterology, 128:A-112, abst 724. 2002).
An enema formulation of ISIS 2302 was used in a clinical trial for the treatment of pouchitis (US Patent Publication 20040162259, see Example 17, Miner et al., Alimnet. Pharmacol. Ther., 19:281-6.2004). Twelve patients with chronic, unremitting pouchitis and a Pouchitis Disease Activity Index (PDAI) of at least 7 were enrolled in the study. PDAI is a clinical score based on a combination of factors including stool frequency, rectal bleeding, fecal urgency, abdominal cramps, fever of greater than 100° F., endoscopic and histologicic scores. Traditionally, active pouchitis is defined as having a PDAI score of at least 7. Patients underwent a 6 week course of nightly administration of a hydroxypropyl methylcellulose enema containing 240 mg of ISIS 2302. Evaluations were performed at baseline and at weeks 3, 6, and 10. The primary endpoint of reduction of PDAI at week 6 was reached (p=0.001) with significant decreases in endoscopy and symptom scores seen as early as week 3, and continuing through week 6, with improvements seen in each of the endoscopy components through week 10. Clinical symptom scores also decreased from baseline to week 6. The enema was well tolerated and there were no serious adverse events in the study. No long term follow-up beyond the week 10 evaluation was performed.
A small randomized, controlled, double-blind escalating dose study of rectally delivered ISIS 2302 for the treatment of mild to moderate ulcerative colitis (SJH van Deventer et al., Gut, 53:1646-51). Patients were treated with one of four daily doses of ISIS 2302 (6, 30, 120 and 240 mg) for 28 days, and the safety and efficacy of the treatment were monitored after 1, 3 and 6 months for improvement in disease activity index (DAI). DAI is a clinical score based on stool frequency, rectal bleeding, endoscopic appearance and investigator's global assessment. None of the patients in the 240 mg/day group or compared with 50% of the patients in the placebo group required additional surgical or medical intervention over baseline during the six months of the study. The results showed promising acute and long term benefits; however, the results required verification in a larger clinical trial to be conclusive.
Despite a number of possible interventions for the treatment of IBD, none of them are fully satisfactory due to limited efficacy, undesirable side effects or both. There clearly remains an unmet need for effective treatment of IBD, preferably treatments that produce sustained effects due to the chronic nature of the disease.